Instruments which analyze small particles suspended in liquids in order to provide parameter distribution data or classification of the particle types are of practical importance in fields which include microbiology, medicine, and drinking/wastewater processing. Some of these instruments form images of a volume of liquid along with any particles contained within this volume and analyze these images to obtain the required parameters. In order to analyze a reasonable number of particles in an acceptable time period, the liquid containing the particles is flowed through an optical sample cell at sufficient speed so that successive images (frames) taken by the camera are of fresh material. Since the particles are in motion, each frame must be collected sufficiently quickly so that motion during image capture is controlled to an acceptable level. Rapid image collection requires an adequately fast detector and adequate illumination intensity to achieve an acceptable signal to noise ratio with these detectors.
Detection systems most often employ the use of computers or powerful processor-based systems coupled to one or more charge coupled devices (CCD). CCD or pixel arrays of detecting elements, which detect the presence of one or more particles projected upon a portion of the array of charge coupled elements. Often thousands of frames of information are collected. Within a single frame more than a single particle may be detected; therefore, the software is programmed to find clusters of pixels, indicating the presence of a particle, and to determine a number of pixels, or a pixel total, for the cluster. Some software can determine instances where portions of particles overlap and determine the size of each particle.
Many prior art systems exist for detecting the presence of particles or size of particles in a fluid, such as a supply of potable water. For example, U.S. Pat. No. 5,438,408 entitled “Measuring Device and Method for the Determination of Particle Size Distributions by Scattered Light Measurements” discloses the use of a CCD camera. U.S. Pat. No. 6,061,130 entitled “Apparatus for Determining the Particle Size Distribution of a Mixture” discloses an apparatus that includes a CCD matrix. By identifying particles by predetermined parameters, such as diameter or cross-sectional area, such systems can ascertain the presence or absence of unwanted harmful bacteria in a water sample if a range of diameters of the bacteria is known.
Some of these systems have also been known to be useful in analyzing other fluids such as blood and blood products. Such systems in the area of microbiology and medicine use fluorescent imaging for particle detection and analysis in blood products or other fluids.
Fluorescence is re-emission of light by certain molecules, fluorophores, as they revert to the ground state following excitation by an optical source. The emitted wavelength spectrum is normally longer than the excitation/absorption wavelength spectrum and is characteristic of the molecule being excited. The intensity of fluorescent emission depends on intensity of the excitation light. For the small objects of interest in micro-biological analysis, fluorescence intensity is normally small and high intensity illumination combined with sensitive signal detection is employed.
Fluorescence is commonly employed in microbiological analysis for identification of target entities through detection of naturally occurring fluorophores which they contain. If a target does not contain natural fluorophores, a fluorescent stain or tag may be employed. Different stains are used to selectively label the entity (or parts of the entity) of interest.
In image gathering by static fluorescent microscopy, static samples, positioned on a microscope slide, are illuminated with an excitation light which will be absorbed by the fluorophore. The sample is imaged either by eye or by a camera at a wavelength band corresponding to the emission wavelength. The excitation light is excluded by wavelength selective filtering. Since the target is static, the time taken to acquire the image may be as long as required. The microscope may subsequently be adjusted to obtain a non-fluorescent image of the same target. If this process is carried out manually, scanning a large number of entities is time consuming.
Automatic scanning instruments may consist of microscopes provided with stepwise movement of a slide under computer control. Successive locations on the slide are illuminated, examined for fluorescence and imaged. For example, a microorganism detecting apparatus provided in U.S. Pat. No. 6,122,396 in the names of King, et al. granted on Sep. 19, 2000, comprises a fluorescence microscope and a motor-driven stage assembly for moving a sample slide underneath an imaging subsystem and above an illumination subsystem.
In automatic fluorescent flow cytometry systems, images are not collected but individual detectors are used to detect and measure, in one or more wavelength bands, the total fluorescent emission of target particles suspended in a flowing liquid. This information along with additional, non-fluorescent, morphology measurements, obtained by measuring scattered signals, is used to examine and classify selectively tagged targets commonly consisting of cells or cell fragments.
For example, an optical analytical apparatus is described in U.S. Pat. No. 4,979,824 granted to Mathies et al. on Dec. 25, 1990. This apparatus is based on a flow cytometry system and utilizes a spatial filter to define a small probe volume that allows for detection of individual fluorescent particles and molecules. Laser power and exposure time of the sample are selected to enhance signal-to-noise ratio. Real-time detection of photon bursts from fluorescent particles is used to provide the number, location or concentration of the particles.
For particle imaging systems employing a constant fluid flow, exposure time of a single frame is limited by the effect of streaking when particles within the fluid change their positions significantly during exposure. In order to obtain statistically significant results, it is required that a large number of particles be analyzed in a reasonable period of time, so rates of 1000 to 10,000 particles/sec or more are desirable. High throughput of an imaging system is associated with a high velocity of the fluid, which causes streaking, undesirable and limiting the exposure. The shorter exposure time requires more intense illumination, which, in turn, can adversely affect the sample, especially in applications related to microbiology and medicine.
There are partial solutions known in the art for extending exposure time by electronically compensating for an accurately predetermined object velocity, e.g. the time-delay integration technique used in U.S. Pat. No. 6,975,400 issued to Ortyn, et al. on Dec. 13, 2005.
It is known in the art, that fluorescent imaging requires relatively long exposure thus slowing down flow velocity in a particle imaging system. Moreover, it is advantageous to collect both fluorescent and non-fluorescent images of the same sample, which further decelerates the fluid flow.
An object of the present invention is to provide a method and a system for automatic imaging of particles in a fluid, allowing for long exposure of a sample while providing a high rate of sampling.